Nematode infection in liver of the fish Gymnotus inaequilabiatus (Gymnotiformes: Gymnotidae) from the Pantanal Region in Brazil: pathobiology and inflammatory response

Abstract

Background: A survey on endoparasitic helminths from freshwater fishes in the Pantanal Region (Mato Grosso do Sul, Brazil) revealed the occurrence of third-larval stage of the nematode Brevimulticaecum sp. (Heterocheilidae) in most organs of Gymnotus inaequilabiatus (Gymnotidae) also known by the local name tuvira. The aim of the present study was to examine Brevimulticaecum sp.-infected tuvira liver at the ultrastructural level and clarify the nature of granulomas and the cellular elements involved in the immune response to nematode larvae.

Methods: Thirty-eight adult specimens of tuvira from Porto Morrinho, were acquired in January and March 2016. Infected and uninfected liver tissues were fixed and prepared for histological and ultrastructure investigations.

Results: The prevalence of infection of tuvira liver by the nematode larvae was 95 %, with an intensity of infection ranging from 4 to 343 larvae (mean ± SD: 55.31 ± 73.94 larvae per liver). In livers with high numbers of nematode larvae, almost entire hepatic tissue was occupied by the parasites. Hepatocytes showed slight to mild degenerative changes and accumulation of pigments. Parasite larvae were surrounded by round to oval granulomas, the result of focal host tissue response to the infection. Each granuloma was typically formed by three concentric layers: an outer layer of fibrous connective tissue with thin elongated fibroblasts; a middle layer of mast cells entrapped in a thin fibroblast-connective mesh; and an inner layer of densely packed epithelioid cells, displaying numerous desmosomes between each other. Numerous macrophage aggregates occurred in the granulomas and in the parenchyma.

Conclusions: Our results in tuvira showed that the larvae were efficiently sequestered within the granulomas, most of the inflammatory components were confined within the thickness of the granuloma, and the parenchyma was relatively free of immune cells and without fibrosis. Presumably this focal encapsulation of the parasites permits uninfected portions of liver to maintain its functions and allows the survival of the host.

Keywords: Fish immune response; Hepatic granuloma; Histopathology; Nematode larvae.

Fig. 1

a Photograph during necropsy; several encysted larvae (arrows) of the nematode Brevimulticaecum sp. are visible on liver of Gymnotus inaequilabiatus. b Very heavily infected liver shown during necropsy examination; the larva appeared as single (arrows) or in clusters (arrowheads) beneath the serosa. c Small piece of fixed liver free of larvae; smooth surface of organ is appreciable. d Cluster of Brevimulticaecum sp. third-stage larva in cysts (arrows) covered by serosa are evident. Note the difference between surfaces of liver in (c and d). e Encysted larva on surface of liver (arrow), and numerous larvae inside the cyst attached to the liver by a peduncle (curved arrow). Scale-bars: b, 1 mm; c, 2 mm; d, 2 mm; e, 1.5 mm

Fig. 2

a Sagittal section of heavily infected liver; the cysts are round to oval in shape and likely occupy the whole hepatic surface (hematoxylin & eosin). b Three different granulomatous reactions are apparent. The granuloma on the right (arrowhead) lacks an evident collagenous encapsulation, which is present in the other two granulomas (arrow and curved arrow). The presence of necrotic material exfoliating form the inner layer is clearly appreciable in the granuloma in the middle of the figure (curved arrow) (Masson’s Trichrome). c Inside the cyst the nematode (arrows) is immersed in an Alcian Blue positive fluid (asterisk) (Alcian Blue-PAS). d Stratification of granuloma is visible, inner layer formed by epithelioid cells (arrows), middle layer is constituted by mast cells (curved arrows), outer layer with connective fibres (arrowheads) and elongated fibroblasts (Masson’s Trichrome). e Infected liver; intense degranulation of the mast cells (arrows) and macrophage aggregates (arrowheads) are evident (Masson’s Trichrome). f Sagittal section of the infected liver; numerous macrophage aggregates are evident (arrows) (Giemsa). g Sinusoids dilation (arrowheads) and liver hydropic degeneration are appreciable, brownish pigments are visible inside the liver cells (arrows) (Giemsa). Scale-bars: a, 500 μm; b, f, 200 μm; c, 50 μm; d, e, 10 μm; g, 10 μm

Fig. 3

a Transmission electron microscope micrograph showing three layers which formed the granuloma; inner layer formed by epithelioid cells (arrows), mast cells (curved arrows) of the middle layer, fibroblasts (thick arrows) and connective fibres (arrowheads) of the outer layers. b The epithelioid cells (arrows) surrounding the nematode larva (asterisk); note the foamy aspect of the epithelioid cells. c Numerous desmosomes (arrows) between epithelioid cells are visible. d Interdigitation (curved arrows) and desmosomes (arrows) between the epithelioid cells. Scale-bars: a, 3.64 μm; b, 3.57 μm; c, 0.26 μm; d, 0.25 μm

Fig. 4

a Micrograph showing the outer layer of the granuloma, abundant collagenous fibres (arrowheads) and fibroblasts (arrows). b High magnification of the middle layer; note many mast cells (curved arrows) and electron-dense granules in their cytoplasm. c Eccentric nucleus (arrow) and several electron-dense granules of a mast cells. d A neutrophil (arrow) surrounded with blood cells within the lumen of a hepatic sinusoid. e Macrophage aggregates (MAs) formed by groups of large oval to round cells (arrows) inside liver parenchyma. f A hepatocyte (arrow) appeared as large polyhedral cell in close proximity to a small vessel (arrowhead). Scale-bars: a, 3.33 μm; b, 5.00 μm; c, 1.30 μm; d, 1.75 μm; e, 4.36 μm; f, 1.55 μm